Proteins and nucleic acids employ only a small fraction of the available functionality. There is considerable current interest in modifying proteins and nucleic acids to diversify their functionality. Molecular evolution efforts include in vitro diversification of a starting molecule into related variants from which desired molecules are chosen. Methods used to generate diversity in nucleic acid and protein libraries include whole genome mutagenesis (Hart et al., Amer. Chem. Soc. (1999), 121:9887-9888), random cassette mutagenesis (Reidhaar-Olson et al., Meth. Enzymol. (1991), 208:564-86), error-prone PCR (Caldwell, et al. (1992), PCR Methods Applic. (1992), 2: 28-33), and DNA shuffling using homologous recombination (Stemmer (1994) Nature (1994), 370:389-391). After diversification, molecules with novel or enhanced properties can be selected.
Methods that enable recombination to take place at defined sites without sequence homology have been described. For example, it is possible to recombine unrelated protein-encoding genes by using synthetic oligonucleotides to encode each desired crossover (O'Maille (2002) J. Mol. Biol. 321:677-91; and Tsuji (2001) Nuc. Acids Res. 29:E97). Although this strategy can result in a high likelihood of preserving function after diversification, many fewer sites of recombination, and therefore, fewer novel structures are accessible than if crossover sites were randomly generated. Alternatively, methods allowing a single nonhomologous crossover of two protein-encoding genes have been developed (Sieber (2001) Nat. Biotechnol. 19:456-60; and Ostermeier (1999) Nat. Biotechnol 17:1205-9), and additional nonhomologous recombination events can be obtained by fragmenting and homologously recombining the resulting genes (Lutz (2001) Proc. Natl. Acad. Sci. USA 98:11248-5317). Despite efforts to enhance the number of crossovers obtained, existing methods for diversifying proteins by nonhomologous recombination have thus far yielded only modest numbers of recombination events (three or fewer per 500 base pair (bp) in protein-encoding sequences, with even fewer crossovers (one to two per 500 bp) among sequences encoding active proteins (Kawarasaki (2003) Nuc. Acids Res. 31:e12618).
While laboratories have explored various aspects of continuous evolution, no generalizable, continuous directed evolution system has been reported. For example, the Joyce laboratory has recently reported continuous evolution of RNA ligase ribozymes. However, their system cannot be generalized to evolve protein functionalities, and is sharply limited in the types of ribozyme activity that can be selected for. (Wright M C, Joyce J F (1997). Science 276: 614-617). The Loeb laboratory created an error-prone polymerase I mutant that selectively diversifies sequences downstream of the colE1 plasmid origin and used it to evolve beta lactamase to resist azneotram (Camps M, Naukkarinen J, Johnson B P, Loeb L A (2003). Proc. Natl. Acad. Sci. USA 100: 9727-9732). However, reliable continuous mutagenesis was not achieved, selections were performed in slow, discrete rounds, and the entire cell was the object of the selection rather than the construct encoding the resistance gene alone. The Bamford laboratory cloned the beta lactamase gene into the genome of the carrier-state RNA virus phi6 in P. aeruginosa. (Makeyev E B, Bamford D H (2004). J. Virol. 78: 2114-2120). Error-prone replication of the RNA virus genome and the beta lactamase gene resulted in a library which was selected for cefotaxime resistance over four passages. While mutagenesis was continuous, the passaging and therefore selection was slow and discrete, the library was not transmitted from cell to cell, and the cell was the object of selection. In addition, the Church laboratory has recently (Wang H H et al., (2009). Nature 460: 894-898) described a MAGE system, which automates the iterative transformation of bacterial cells with nucleic acids. However, there is no intrinsic means of screening or selecting for a desired function; that is, functional mutants are not selected for without discrete intervention. As such, MAGE represents an important advance over traditional directed evolution techniques, but is not truly continuous and remains considerably slower and more limited in library size than the present invention.
Accordingly, a need exists for a continuous, generalizable, effective method of evolving nucleic acids and proteins.